AVI has another paper using their Acp knockdown in E. coli to assess ability of some modified Morpholinos to enter bacterial cells.

Mellbye BL, Weller DD, Hassinger JN, Reeves MD, Lovejoy CE, Iversen PL, Geller BL. Cationic phosphorodiamidate morpholino oligomers efficiently prevent growth of Escherichia coli in vitro and in vivo. J Antimicrob Chemother. 2009 Nov 1. [Epub ahead of print]

The bottom line: their arginine-rich peptide-conjugated Morpholinos worked better than the new modifications they tested. 

However, their approach is interesting and I think the test was worthwhile.  In an attempt to improve uptake into the bacterial cells, they modified the phosphorodiamidate linkage of the Morpholino oligos.  Normally, there is a dimethylamine attached to the phosphorus.  They substituted this with larger groups, either a piperazine or a (6-guanidinohexanoyl)piperazine.  Of these two new backbone modifications, the (6-guanidinohexanoyl)piperazine enhanced delivery and the piperazine alone didn't do much. 

I would like to see how a knockdown in a mouse with the (6-guanidinohexanoyl)piperazine-modified Morpholinos compares with the same target knocked down with an arginine-rich peptide-conjugated Morpholino.  Assuming that the oligos are being assembled from modified subunits that contain some or all of the new substituants on the phosphorus, you might be able to assemble such a backbone-modified oligo more rapidly than if you had to synthesize a Morpholino and then attach a peptide.  That's useful for a business like Gene Tools, where we are constantly making new Morpholino sequencs, but less important for a pharma-focused company which generally makes larger batches of fewer sequences.  I wouldn't be surprised to see this new structure fade away without more publications -- they didn't beat their (RXR)4XB peptides for delivery efficacy into the Gram negative bacteria.  Nice try with some intriguing chemistry!

Nice publications appearing today!

Morpholino targeting miRNA:
Zeng L, Carter AD, Childs SJ. miR-145 directs intestinal maturation in zebrafish. Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17793-8. Epub 2009 Oct 5.

---

---

I wrote a short lay-audience essay on exon-skipping oligos and Duchenne muscular dystrophy for Marie's blog, GenMedLabMom; see the Oct. 7th 2009 post:   http://genmedlabmom.blogspot.com/

siRNA can decrease the expression level of a protein, but some protein remains.  I occasionally point out that Morpholinos can sometimes knock down expression of a protein to a level undetectable by Western blot.  We've seen this off and on reported in papers, but rarely so explicitly as in this conference abstract.

Invest Ophthalmol Vis Sci 2009;50: E-Abstract 4361.

Morpholino Knockdown of -Crystallin in the Zebrafish (Danio rerio)

M. Posner¹, N. Meyers¹ and J. O. Liang²
http://abstracts.iovs.org/cgi/content/abstract/50/5/4361

Moulton HM, Wu B, Jearawiriyapaisarn N, Sazani P, Lu QL, Kole R.
Peptide-morpholino conjugate: a promising therapeutic for Duchenne muscular dystrophy.
Ann N Y Acad Sci. 2009 Sep;1175:55-60.

http://www.ncbi.nlm.nih.gov/pubmed/19796077

This is a paper Hong submitted in combination with presenting at a conference, distilling the main points of several previous research papers.  It's not the very latest research result, but rewriting and combining several recent projects produces a readable story, like a brief review.

Here is a response I wrote for BioForum (http://www.protocol-online.org/forums/) in a discussion of how to determine whether an miRNA targeted a particular mRNA.  One of the regular forum participants, miRNA man, posted a question about the specificity of Morpholinos targeting miRNA response elements (RNA sequences, generally in the 3'-UTR of an mRNA, where miRNAs bind and suppress translation of the mRNA).  The following discussion addresses controls for the specificity of Morpholino target protectors, which are oligos that protect the miRNA response elements.

Hi miRNA man,

You are right, it takes careful controls to show specificity of a Morpholino-RNA interaction.  The Morpholino oligos are more specific than most other antisense types (due to low per-base affinity and consequent long complementarity requirement), but off-target binding sometimes happens enough to cause misleading results.  The problem of proving specificity for an oligo protecting an miRNA target site hasn't been thoroughly worked out as far as I know.  The last paragraph of this discussion specifically addresses a new technique for controlling for the specificity of target protectors.  Many of these techniques require careful control of oligo dose, so the solution concentrations of the Morpholinos should be confirmed by UV spectrometry prior to dosing.

For translation blocking or splice modifying Morpholinos, a common method of controlling for specificity is to use two distinct Morpholinos with non-overlapping target sites.  If the oligos are used in separate experiments and produce the same change, this supports the hypothesis that the observed change is due to interactions with the targeted RNA and not due to unintended Morpholino-RNA interactions (since a random RNA binding to one oligo would be unlikely to have sufficient complementarity with the second oligo to cause the same change with each).  Note that the two oligos will usually have somewhat different efficacies, and the minimum dose that causes the change will need to be independently determined.   A second specificity test using the same pair of oligos involves coinjecting the pair of oligos and looking for dose synergy -- If a phenotype elicited by a single Morpholino can be reproduced by a coinjection where the sum of the concentrations of the two coinjected oligos is considerably less than the concentration of the original single oligo, this supports the hypothesis that the phenotype was specific.

Back to blocking miRNA activity with Morpholinos.  First, when targeting the miRNA itself, one Morpholino is used which targets the guide strand (and usually overlaps the guide-Dicer site) and another oligo is used targeting the star strand (usually star-Dicer targeted).  While the star-targeted oligo cannot bind to the miRNA on RISC (assuming the star strand doesn't load into RISC), the star-Dicer oligo can inhibit maturation of the miRNA (by protecting the Dicer site from cleavage).  This means that the two oligos can be used as a specificity control pair; while the star oligo might produce a slightly weaker phenotype, the ability to mostly phenocopy the guide oligo lends support to the specificity hypothesis.  However, this is complicated by the close sequence similarity within miRNA families; Morpholinos can generally still bind well enough to have some biological effect even if there is a mispair or two between the oligo and its target.  To target one member of a family, I try to design oligos overlapping more loop sequence, since within a miRNA family the loop region is generally far less conserved than the guide or star sequences.  The star-strand control was first proposed by Wigard Kloosterman:  Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH.  Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development.  PLoS Biol. 2007 Jul 24;5(8):e203 [Epub ahead of print]

Next, targeting miRNA target sites.  There are not very many reports of target-protecting with Morpholinos in the literature, so a consensus has not been reached about the techniques for specificity control.  One possibility is to use two target-protecting oligos, each covering half of the miRNA target site and extending in different directions across the flanking sequence.  First one or the other of these is used and the concentrations determined that produce a weak phenotype.  When they are both used together, the phenotype should strengthen considerably, showing a dose-synergy effect IF the single-oligo phenotypes are caused by blocking the intended miRNA target site.  A good confirmation is to drop the concentration of the coinjected oligos and determine whether they are still producing the phenotype of interest at a summed concentration below that required for a single oligo.  This is not a well-vetted technique and I've not seen it published yet, but it is an approach that parallels the standard techniques used for other classes of Morpholino targets.

This paper reports doses up to 3g/kg in mice, resulting in dystrophin exon-skipping and considerable dystrophin production in cardiac muscle.  The creatures were 0.3% oligo by weight just after the retro-orbital injections -- and the mice survived!  If a Morpholino sequence is non-toxic, amazing amounts can be tolerated.

Wu B, Lu P, Benrashid E, Malik S, Ashar J, Doran TJ, Lu QL. Dose-dependent restoration of dystrophin expression in cardiac muscle of dystrophic mice by systemically delivered morpholino. Gene Ther. 2009 Sep 17. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/19759562

The folks at Phalanx Biotech have put a copy of our poster, presented during the Gene Tools roundtable at the Society for Developmental Biology meeting 2009, on the web.  This describes some microarray work undertaken to determine which of our negative control oligos causes less off-target gene modulation.  The bottom line is that the 25-base length random control oligo mixture (N-25) is a quieter control than the standard control oligo.

http://www.phalanxbiotech.com/Posters/SDB_GnT_PHX_Poster.pdf

I'm back from Europe, jet-lagged and at my desk with coffee.

Three weeks in Europe and the UK,flights, rail and rental car, nine hotels, France, Switzerland, Scotland and England, two presentations (by my wife Hong in Montpelier and Lausanne), one meeting exhibit (by me at International Society for Developmental Biology), a lab visit (Hong's collaborators Prof. Matthew Wood and Dr. Haifang Yin in Oxford) and all this with my daughters, 9 and 5 years old, in tow and adding color.  Visits to the Louvre, Musée d'Orsay and Musée de l’Orangerie. We watched the east coast of the UK from the railway window as we rode from Edinburgh to London.  We left Heathrow yesterday morning and jetlag will take a day or two to shake off.  A good adventure overall.